Project Summary Mycobacterium tuberculosis (Mtb) has adapted to survive a wide range of assaults?from our immune response to antimicrobial therapeutics?intended to eradicate the organism. However, the molecular switches that enable Mtb to endure these stresses, to slow replication or to become dormant as a latent tuberculosis (TB) infection are not known. Emerging studies on the molecular underpinnings of stress survival generally point to a major role for toxin-antitoxin (TA) systems, which are operons comprising adjacent genes encoding two small proteins, a toxin and its cognate antitoxin that inhibits toxin activity in the TA protein-protein complex. However, several bottlenecks have impeded progress toward rigorous testing of this provocative association. This proposal enlists a strong multidisciplinary team with expertise in all core components of the proposed work?RNA-seq, TA systems, Mtb biology/physiology and bioinformatics/computational biology. The genome-scale approach developed in the PI?s laboratory, 5? RNA-seq, will be used to overcome these obstacles as they apply to the eleven-member MazE (antitoxin) ? MazF (toxin) family in Mtb. 5? RNA-seq will facilitate comprehensive detection of MazF targets in the Mtb transcriptome under unstressed conditions or after exposure to stresses that are relevant to latent TB infection. Finally, the impact of MazF toxins on the Mtb proteome will be investigated. Collectively, these approaches will identify the environmental signals that trigger toxin activation in Mtb, provide an accurate snapshot of RNAs targeted by MazF toxins under these metabolic states, and reveal clues to how toxin-mediated RNA cleavage alters Mtb physiology. These goals align well with ?Priority 1: Improve Fundamental Knowledge of TB? of the five components of the NIAID Strategic Plan for Tuberculosis Research released in September 2018.